Our long-term goal is to promote transplant tolerance in pediatric liver transplant recipients so that immunosuppression and the associated morbidity and mortality risks can be eliminated. Transplant tolerance can spontaneously develop and be identified through immunosuppression withdrawal. However, data consistently show that rate of spontaneous tolerance is low early after transplantation and increases slowly over time as multiple toxicities accumulate. Children face a particularly onerous burden of immunosuppression, extending over a long lifespan. Therefore new therapies that facilitate tolerance induction are much needed. Research in the past 20 years has demonstrated that regulatory T cells are essential for immune tolerance to self-antigens and can be therapeutically harnessed to induce transplant tolerance. Recently concluded phase I clinical trials in graft versus host disease and an ongoing clinical trial in type 1 diabetes demonstrate that regulatory T cell therapy is safe and potentially efficacious in suppressing alloimmune responses. Here we propose to conduct an open label, phase I/II, multi-center clinical trial to determine the safety and potential efficacy of a single infusion of autologous, polyclonally expanded, regulatory T cells to induce tolerance in non- tolerant pediatric liver transplant recipients. We will also perform mechanistic studies using peripheral blood and biopsy samples collected from trial participants to improve our understanding of transplantation tolerance and the impact of regulatory T cells on alloimmune responses in humans. We have established a collaborative network of six clinical centers and four mechanistic cores that have unique and relevant expertise as well as many pre-existing collaborations to conduct this trial. We plan to enroll 25 children 2 to 6 years after liver transplantation and identify non-tolerant patients through highly supervised gradual immunosuppression withdrawal. Twelve non-tolerant patients will be stabilized with immunosuppression for 6 to 12 months before receiving an infusion of autologous expanded regulatory T cells. These participants will undergo second attempt of immunosuppression withdrawal to determine whether regulatory T cell therapy can induce tolerance. Global gene expression profiling of blood and biopsy samples, multi-parameter immunohistology of liver tissue, and multiplex plasma cytokine and chemokine analysis will be used to identify biomarkers of tolerance, differences in immune responses between tolerant and non-tolerant patients, and impact of regulatory T cells on the immune responses. This proposal is strongly resonates with the mission of NIAID and CTOT-C to promote understanding and reduce immune-mediated morbidity and mortality in vulnerable pediatric transplant recipients.